One of the major foci of research in our laboratory is the determination of mechanisms underlying neuromodulation and plasticity and the effects of alcohol and other drugs of abuse on these neuronal functions. In particular, we are interested in the function of the striatum, a brain region involved in action control, action selection and learning. [unreadable] We have continued our studies of long-term synaptic depression (LTD) a form of long-lasting synaptic plasticity that is thought to contribute to striatal-based learning and memory. Our recent studies have focued on LTD at inhibitory synapses in striatum that use the neurotransmitter gamma-aminobutyric acid (GABA), and comparison to LTD at excitatory synapses. LTD at GABAergic synapses can be induced by low frequency afferent stimulation (1 Hz), while much higher stimulation frequencies are required for LTD induction at glutmatergic synapses. In addition, induction of LTD by activation of presynaptic CB1 receptors requires strong synaptic activation at glutamatergic synapses, while explicit synaptic activation is not required for LTD at GABAergic synapses. Finally, striatal GABAergic synaptic transmission is more sensitive to inhibition by CB1 agonists in comparison to striatal glutamatergic transmission. Thus, striatal GABAergic synapses are more sensitive targets for cannabinoid and endocannabinoid modulation and synaptic plasticity than glutamatergic synapses, most likely due to the fact that the number of CB1 receptors is higher on GABAergic than on glutamatergic presynaptic terminals in this brain region. These findings have important implications for our understanding of how cannabinoid drugs and activation of endocannabinoid signaling affect striatal function, We are presently comparing mice with different levels of CB1 receptor expression to determine if modulation and plasticity at GABAergic or glutamatergic synapses show differential sensitivity to receptor expression level. We can then compare these findings to behavioral data generated in these animals by the Section on In Vivo Neural Function (SIVNF) to gain a better understanding of how cannabinoidergic transmission at different striatal synapses contributes to striatal-based behaviors, including habit formation. We are also exploring techniques for activation of selected GABAergic neurons within the striatum in order to determine which of the several types of GABAergic synapses in this brain region are sensitive to cannabinoid and endocannabinoid actions. [unreadable] In collaboration with both SIVNF and the Section on Behavioral Science and Genetics (SBSG) we have undertaken experiments designed to determine the changes in synaptic transmission that underlie learning and memory. In one study, mice were trained on a skill task using the accelerating rotarod, and striatal brain slices were examined from animals at different stages of training in comparison to control mouse striatal slices. Exposure to rotarod training produced changes in excitability of striatal medium spiny projection neurons (MSNs) as well as enhanced glutamatergic synaptic transmission onto these neurons. The enhanced excitability was related to exposure to the training apparatus, while the synaptic potentiation was more closely related to skill learning. Interestingly, changes in the associative striatum were observed early in training, while changes were more robust in the sensorimotor striatum with extensive training. This finding fits well with a growing body of literature suggesting that early phases of action learning where acquisition is rapid and behavior is flexible involve associative circuitry. A slower phase of action learning that leads to inflexible behavior is thought to involve sensorimotor circuitry. This work has important implications for the study of learning in general, and may also relate to habit formation and addiction, as the striatum is implicated in these behaviors. By understanding the cellular and molecular changes that underlie action learning we hope to gain a better understanding of neuroadaptations that take place during addiction, alcohol abuse and alcoholism. [unreadable] In collaboration with SBSG we have examined transmission and plasticity at glutamatergic synapses in mice that lack the NR2B subunit of the NMDA-type glutamate receptor, created by Dr. Eric Delpire of Vanderbilt University as part of the NIAAA-sponsored INIA alcohol research consortium. The NR2B subunit is involved in synaptic plasticity underlying learning and memory, such as long-term potentiation (LTP). Expression and localization of NR2B is altered by chronic ethanol exposure. We have used mice in which NR2B is specifically absent in the hippocampal and neocortical brain regions. LTP at glutamatergic synapses in hippocampus is strongly impaired in mice lacking NR2B. The synaptic responses mediated by the NMDA receptor are shorter in duration at these synapses, and this change may underlie the LTP deficit. Work in SBSG shows that mice lacking hippocampal and cortical NR2B are deficient in learning tasks involving these brain regions. We are now initiating studies of chronic alcohol effects on these mice. These studies are being performed in collaboration with other INIA investigators. Use of these mice should help us to understand the role of NR2B-containing NMDA receptors in neuroadaptations brought about by chronic alcohol exposure and withdrawal.[unreadable] We are also interested in the molecular basis of ethanol actions on the brain. We have studied the 5-HT3 receptor for the neurotransmitter 5-hydroxytryptamine (aka serotonin), a protein whose function is enhanced by ethanol. The 5-HT3 receptor can exist as a homopentamer if only the A subunit protein is present. However, the 5-HT3B subunit also contributes to formation of heteropentameric receptors that are insensitive to potentiation by ethanol. At present, little is known about how ethanol affects the function of 5-HT3 receptors in mammalian brain. Of relevance to this question is which subunits contribute to the formation of brain 5-HT3 receptors, and this is not known. Working with Paul Davies of Tufts University we are examining the subunit composition, function and alcohol sensitivity of brain 5-HT3 receptors using a combination of electrophysiological recording, pharmacological analysis and RNA measurement. In preliminary experiments we have measured mRNA for both the 5-HT3A and B subunits in tissue microdissected from both basolateral amygdala (BLA) and hippocampus. The A subunit appears to be much more abundant than B in these brain regions. Previous work indicated that 5-HT3 receptors are mainly expressed by GABAergic interneurons in the brain. Thus, we identified different types of interneurons using mice in which the expression of green fluorescent protein (GFP) is driven by the promoter for glutamic acid decarboxylase 65 (GAD65). Electrophysiological recordings from isolated neurons indicated the presence of 5-HT3-mediated current in a subset of interneurons. No evidence of functional receptors was observed in the majority of interneurons or in projection neurons in either brain region. In mechanically-isolated neurons that retain intact GABAergic synapses, 5-HT3 activation enhances GABA release onto projection neurons, with no apparent postsynaptic effect. This effect was consistently observed in neurons from both BLA and hippocampus. Thus, the most consistent action of this receptor is to boost inhibitory GABAergic transmission. Preliminary results indicate that ethanol can potentiate this presynaptic 5-HT3 receptor action in some, but not all, neurons. We are currently characterizing the pharmacology and ethanol sensitivity of the presynaptic and postsynaptic 5-HT3 receptors.